Location of ligandin's four functions on the protein molecule: The peptides generated by limited proteolysis of ligandin with subtilisin will be separated by high pressure liquid chromatography. Conditions such as pH and composition of buffer, elution gradient will be optimized for maximal resolution. The peptide containing fractions will then be assayed for ligandins four functions. (GSH-S-transferase, steroid isomerase, bilirubin binding and immunlogical reactivity). Isolation of azodye bound ligandin and determination of its binding, catalytic and immunological properties: Rats would be fed 3'-Me-DAB for 8 weeks. The ligandin will be isolated from the livers of these rats by chromatography of cytosol on TEAE cellulose, sephadex G-75 and QAE A50 sephadex columns. We will then determine ligandins catalytic binding and immunological properties by assaying for GSH-S-transferase and steroid isomerase activities, bilirubin and sulfobromophthalein binding ability and immunological reactivity (by Mancini's method). Purification of mRNA for ligandin and development of cDNA probe: The mRNA for ligandin would be purified further by preparative gel electrophoresis and rot hybridizations. It would then be reverse transcribed in presence of C14 deoxy CTP to obtain cDNA. The cDNA preparation would be purified on sucrose gradients.